Parentage testing of racing camels (Camelus dromedarius) using microsatellite DNA typing

The camel racing industry would have added value in being able to assign parentage with high certainty. This study was aimed at assessing and applying microsatellite multiplexes to construct a parentage testing system for camels. An efficient system of 17 loci from 700 camel samples was used to construct a database of unrelated adults. Based on this, we estimated measures of polymorphism among the markers. In three multiplex reactions, we detected a total of 224 alleles, with 5–23 alleles/locus (mean = 13.18 ± 6.95 SD) and an average heterozygosity (H_E) of 0.54 (range 0.032–0.905). The total parentage exclusion probability was 0.99999 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.9999 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. We used 15 juveniles for parentage testing, as well as 17 sires (bull camels) and 21 dams (cows). In the case of parentage assignment, the microsatellite panel assigned all 15 offspring parentage with high confidence. Overall, these findings offer a set of microsatellite markers that are easy, simple and highly informative for parentage testing in camels.     

Parentage testing in alpacas (Vicugna pacos) using semi-automated fluorescent multiplex PCRs with 10 microsatellite markers

The aim of this study was to assess and apply a microsatellite multiplex system for parentage determination in alpacas. An approach for parentage testing based on 10 microsatellites was evaluated in a population of 329 unrelated alpacas from different geographical zones in Perú. All microsatellite markers, which amplified in two multiplex reactions, were highly polymorphic with a mean of 14.5 alleles per locus (six to 28 alleles per locus) and an average expected heterozygosity ( H _E) of 0.8185 (range of 0.698–0.946). The total parentage exclusion probability was 0.999456 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.999991 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. In a case test of parentage assignment, the microsatellite panel assigned 38 (from 45 cases) offspring parentage to 10 sires with LOD scores ranging from 2.19 × 10⁺¹³ to 1.34 × 10⁺¹⁵ and Δ values ranging from 2.80 × 10⁺¹² to 1.34 × 10⁺¹⁵ with an estimated pedigree error rate of 15.5%. The performance of this multiplex panel of markers suggests that it will be useful in parentage testing of alpacas.     

DNA-based parentage verification in two Australian goat herds

This study aimed to evaluate a set of DNA markers for their effectiveness in parentage inference, to quantify the level of pedigree errors in Australian Angora and Cashmere goat herds using different pedigree recording methods, and to investigate genotype mismatches between parent and offspring. The 14 microsatellite markers evaluated in this study provided a high level of power (probability of exclusion, PE >99.70%) for parentage testing. The extent of PE depended on polymorphic information content (PIC) and number of alleles for each marker. The minimum number of MS markers essential for accurate determination of parentage was 12, when neither parent is known (PE1) and 10, when one parent is known (PE2). In both populations, the error rates of recorded sire and dam pedigree were significant, averaging around 12%. The error rates of sire and dam pedigree varied considerably between the two populations, reflecting management differences on the two properties. Of 14 MS markers, one locus, SRCRSP07, had null alleles present in the heterozygous state. This null allele was revealed by mismatches of genotypes of parent-offspring pairs. Highly significant deviation from Hardy–Weinberg Equilibrium and significant heterozygote deficiency was also observed at this locus.     

A Preliminary Genetic Linkage Map of the Pacific Abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F₁ family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (α = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.     

鳙基于10个微卫星标记的亲子鉴定分析

为开展鳙(Hypophthalmichthys nobilis)家系选育工作,本研究进行了基于微卫星标记的亲子鉴定研究。试验中筛选了10个扩增效率较高的微卫星标记,通过引物荧光修饰,引物结合毛细管电泳分型技术,对鳙48尾亲本及384尾子代进行了基因分型,并计算了等位基因频率和模拟分析和亲子鉴定等分析。结果发现,各位点的等位基因数介于4~13之间,其中9个位点均具有较高的多态性和杂合度(PIC>0.5,He>0.5),研究发现位点的多态性信息含量(PIC)与亲本对排除率(E-PP)存在显著正相关(p<0.01)。模拟分析结果显示,该10个标记预计可用于已知性别的50组亲本(100尾)或未知性别的50尾亲本的鉴定分析(鉴别成功率>95%)。亲子鉴定发现,对试验中2个交配组(每组12对亲本)的鉴别成功率分别为98.96%和100%;且父母本对子代的贡献率存在极显著差异(p<0.01)。通过累积位点的鉴定分析发现,当标记数为7个和9个时分别能满足试验中12组和24组亲本对应子代的鉴定分析(鉴别率>95%),模拟分析和亲子鉴定分析成功率趋势基本符合。本研究所开发的亲子鉴定技术可为鳙家系选育提供技术支持。     

Validation of microsatellite multiplexes for parentage analysis and species discrimination in two hybridizing species of coral reef fish (Plectropomus spp., Serranidae)

Microsatellites are often considered ideal markers to investigate ecological processes in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leopardus and P. maculatus (Serranidae). Coral trout are important fisheries species throughout the Indo‐Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co‐amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent–offspring relationships in natural populations, with over 99.6% accuracy in parent–offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28–96% of loci. The multiplex PCRs developed here provide a reliable and cost‐effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild populations and aquaculture brood stocks for these closely related fish species.     

Randomly Amplified Polymorphic DNA Variation in Populations of Eastern Australian Koalas, Phascolarctos cinereus

Randomly amplified polymorphic DNA (RAPD) variation in populations of the koala, Phascolarctos cinereus, was investigated, revealing significant differences in the level of diversity between southern and northern regions of eastern Australia. Of the 20 polymorphic RAPD markers identified in koalas, 4-7 were polymorphic in southern populations, while 12-17 were polymorphic in northern populations. Analysis of molecular variance revealed a significant difference in the estimated variance between koalas from northern and those from southern regions (P < 0.001), where populations from the north were greater than twice as variable as their southern cousins. The total genetic diversity observed was attributed to regional differences (30.91%), population differences within a region (11.77%), and differences among individuals within a population (57.32%). For the within-region analyses, a large proportion of the genetic diversity was attributable to individual differences within a population, 80.34% for the north and 91.23% for the south. These results demonstrate that RAPD markers are useful for determining population structure among koalas.     

Genetic variation and population structure of endemic yellow catfish, <Emphasis Type="Italic">Horabagrus</Emphasis><Emphasis Type="Italic">brachysoma</Emphasis> (Bagridae) among three populations of Western Ghat region using RAPD and microsatellite markers

Random amplified polymorphic DNA (RAPD) and microsatellite markers were applied to evaluate the genetic variation in endemic and endangered yellow catfish, Horabagrus brachysoma sampled from three geographic locations of Western Ghat, South India river systems. In RAPD, of 32 10-mer RAPD primers screened initially, 10 were chosen and used in a comparative analysis of H. brachysoma collected from Meenachil, Chalakkudy and Nethravathi River systems. Of the 124 total RAPD fragments amplified, 49 (39.51%) were found to be shared by individuals of all 3 populations. The remaining 75 fragments were found to be polymorphic (60.48%). In microsatellites, six polymorphic microsatellite loci were identified by using primers developed for Pangasius hypophthalmus, Clarias macrocephalus and Clarias gariepinus. The identified loci were confirmed as microsatellite by sequencing after making a clone. The nucleotide sequences of 6 loci were published in NCBI genbank. The number of alleles across the six loci ranged from 4 to 7 and heterozygosities ranged from 0.07 to 0.93. The mean number of alleles and effective number of alleles per locus were 5.00 and 3.314, respectively. The average heterozygosity across all investigated samples was 0.72, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods reported a high degree of gene diversity and genetic distances depicted by UPGMA dendrograms among the populations of H. brachysoma.     

DNA microsatellite analysis for parentage control in Austrian pigs.

We present an efficient parentage control for pigs based on ten polymorphic microsatellite markers analyzed in a single PCR reaction. Assuming one known parent ("paternity control"), combined exclusion probabilities (CEPs) ranged from 99.18% (Landrace), 99.74% (Piétrain) to 99.76% (Large White) for the most important Austrian breeds. Assuming a known parent-pair ("parentage control", e.g. a substituted offspring), the CEP of the 10-plex PCR increased to 99.97% (Landrace) and 99.99% (Piétrain and Large White). We developed an additional standby battery of 5 markers, which might be applied in those cases, where the CEP of the 10-plex PCR is not sufficient. Therefore an automated, cost and time reduced genotype analysis for pigs is available.     

The analysis of paternity and maternity in the marine hydrozoan Hydractinia symbiolongicarpus using randomly amplified polymorphic DNA (RAPD) markers

For organisms in which direct observation of mating and subsequent dispersal of offspring and relatives is impossible, patterns of reproductive success and genealogical relationship can only be established using genetic markers. The ideal genetic assay would (1) employ highly polymorphic genetic markers for distinguishing among individuals; (2) use little tissue for analysing early life-history stages; and (3) require minimal investment in time and money for population level studies. From this perspective, DNA polymorphisms revealed by PCR amplification using random ten-base primers [Randomly Amplified Polymorphic DNA (PCR-RAPD) or Arbitrarily Primed DNA (AP-PCR)] have great potential. However, the evidence that RAPD/AP markers are both heritable and can be repeatably amplified remains controversial. This study characterizes patterns of inheritance and polymorphism of RAPD markers in the free-spawning, colonial marine hydrozoan Hydractinia symbiolongicarpus. In all cases, the amplification products were identical among extractions from the same clone. Of 56 primers screened, 13 had sufficient polymorphism and scoreability for an analysis of parentage and higher-order genetic relationships in three matings. These primers generated 156 unique amplification products (putative loci), of which 133 were polymorphic. All but four of these loci were inherited as dominant mendelian markers. Our study suggests that the presence of a marker represents a single allele at a locus; however, what appear to be single null alleles may actually comprise several segregating alleles. When the identity of neither parent was known a priori, inclusion (unique markers present in offspring and only one of the potential parents) proved to be more efficient than exclusion for assigning offspring to parents. The most powerful approach, however, was cluster analysis of all presence/absence information for the marker bands. Clustering avoided the pitfalls caused by the appearance of occasional nonparental bands, and constructed a hierarchical framework that correctly reflected all genealogical relationships.     

DNA-based parentage verification in two Australian goat herds

This study aimed to evaluate a set of DNA markers for their effectiveness in parentage inference, to quantify the level of pedigree errors in Australian Angora and Cashmere goat herds using different pedigree recording methods, and to investigate genotype mismatches between parent and offspring. The 14 microsatellite markers evaluated in this study provided a high level of power (probability of exclusion, PE >99.70%) for parentage testing. The extent of PE depended on polymorphic information content (PIC) and number of alleles for each marker. The minimum number of MS markers essential for accurate determination of parentage was 12, when neither parent is known (PE1) and 10, when one parent is known (PE2). In both populations, the error rates of recorded sire and dam pedigree were significant, averaging around 12%. The error rates of sire and dam pedigree varied considerably between the two populations, reflecting management differences on the two properties. Of 14 MS markers, one locus, SRCRSP07, had null alleles present in the heterozygous state. This null allele was revealed by mismatches of genotypes of parent-offspring pairs. Highly significant deviation from Hardy–Weinberg Equilibrium and significant heterozygote deficiency was also observed at this locus.     

Microsatellite and RAPD polymorphisms in Ontario corn hybrids are related to the commercial sources and maturity ratings

Random-amplified polymorphic DNA (RAPD) and microsatellite markers were used to estimate the genetic relationships among 37 Ontario corn hybrids. Almost all (95%) of the 160 RAPD fragments and all of the 79 microsatellite alleles were polymorphic across the 37 hybrids. Similarity values among the hybrids ranged from 31% to 86% when based on the RAPD data. The similarities based on microsatellite markers ranged from 12% to 77%. The genetic diversity revealed by microsatellite marker analysis was higher than that obtained from RAPD analysis. The similarity matrices for the microsatellite data and the RAPD data were moderately correlated (0.43). Cluster analyses based on either type of marker showed that most of the hybrids from the same company were closely related to each other. Both dendrograms clustered similar pairs or groups of hybrids. A principal component analysis, based on the combined RAPD and microsatellite data, yielded a good separation of the hybrids with Ontario Corn Heat Unit (OCHU) values <2800 from those with OCHU values >2800. Seventeen RAPD markers and 5 microsatellite markers were significantly associated with the OCHU ratings of the hybrids.     

Using blocks of linked single nucleotide polymorphisms as highly polymorphic genetic markers for parentage analysis

Single nucleotide polymorphisms (SNPs) are plentiful in most genomes and amenable to high throughput genotyping, but they are not yet popular for parentage or paternity analysis. The markers are bi-allelic, so individually they contain little information about parentage, and in nonmodel organisms the process of identifying large numbers of unlinked SNPs can be daunting. We explore the possibility of using blocks of between three and 26 linked SNPs as highly polymorphic molecular markers for reconstructing male genotypes in polyandrous organisms with moderate (five offspring) to large (25 offspring) clutches of offspring. Haplotypes are inferred for each block of linked SNPs using the programs Haplore and Phase 2.1. Each multi-SNP haplotype is then treated as a separate allele, producing a highly polymorphic, 'microsatellite-like' marker. A simulation study is performed using haplotype frequencies derived from empirical data sets from Drosophila melanogaster and Mus musculus populations. We find that the markers produced are competitive with microsatellite loci in terms of single parent exclusion probabilities, particularly when using six or more linked SNPs to form a haplotype. These markers contain only modest rates of missing data and genotyping or phasing errors and thus should be seriously considered as molecular markers for parentage analysis, particularly when the study is interested in the functional significance of polymorphisms across the genome.     

Patterns of vegetative growth and gene flow in Rhizopogon vinicolor and R. vesiculosus (Boletales, Basidiomycota)

We have collected sporocarps and tuberculate ectomycorrhizae of both Rhizopogon vinicolor and Rhizopogon vesiculosus from three 50 x 100 m plots located at Mary's Peak in the Oregon Coast Range (USA); linear map distances between plots ranged from c. 1 km to c. 5.5 km. Six and seven previously developed microsatellite markers were used to map the approximate size and distribution of R. vinicolor and R. vesiculosus genets, respectively. Genetic structure within plots was analysed using spatial autocorrelation analyses. No significant clustering of similar genotypes was detected in either species when redundant samples from the same genets were culled from the data sets. In contrast, strong clustering was detected in R. vesiculosus when all samples were analysed, but not in R. vinicolor. These results demonstrate that isolation by distance does not occur in either species at the intraplot sampling scale and that clonal propagation (vegetative growth) is significantly more prevalent in R. vesiculosus than in R. vinicolor. Significant genetic differentiation was detected between some of the plots and appeared greater in the more clonal species R. vesiculosus with Phi(ST) values ranging from 0.010 to 0.078*** than in R. vinicolor with Phi(ST) values ranging from -0.002 to 0.022** (*P < 0.05, **P < 0.01, ***P < 0.001). When tested against the null hypothesis of no relationship between individuals, parentage analysis detected seven likely parent/offspring pairs in R. vinicolor and four in R. vesiculosus (alpha = 0.001). Of these 11 possible parent/offspring pairs, only two R. vinicolor pairs were still supported as parent/offspring when tested against the alternative hypothesis of being full siblings (alpha = 0.05). In the latter two cases, parent and offspring were located at approximately 45 m and 28 m from each other. Challenges to parentage analysis in ectomycorrhizal fungi are discussed.     

Parentage assignment of the mud crab (Scylla paramamosain) based on microsatellite markers

In this study, microsatellite markers were employed to identify the parentage relationship in Scylla paramamosain. The exclusion probability of loci was found to be related with the level of their heterozygosity. When no parent information or only one parent information was available, the exclusion probability ranged from 22.0% to 56.6% and from 41.2% to 73.1%, with the combined exclusion probability for ten loci being 97.0% and 99.8%, respectively. The cumulative assignment success rate was 100% when no parent information was available using seven most informative microsatellite markers. Moreover, the power of the seven microsatellite markers for parentage assignment was tested by a double-blind test, which indicated that 95% of the progeny can be correctly assigned to their parents. This study provided a microsatellite-based approach for parentage assignment in S. paramamosain that will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this important crab species.     

Genetic relationships among wheat genotypes, as revealed by microsatellite markers and pedigree analysis

Genetic relationships among 20 elite wheat genotypes were studied using microsatellite markers and pedigree analysis. A total of 93 polymorphic bands were obtained with 25 microsatellite primer pairs. Coefficient of parentage (COP) values were calculated using parentage information at the expansion level of 5. The pedigree-based similarity (mean 0.115, range 0.00-0.53) was lower than the similarity assessed using microsatellite markers (mean 0.70, range 0.47-0.91). Similarity estimates were used to construct dendrograms by using the unweighted pair-group method with arithmetic averages (UPGMA). Clustering of genotypes in respect of marker-based similarity revealed two groups. Genotype PBW442 diverged and appeared as distinct from all other genotypes in both marker-based and pedigree-based analysis. The correlation of COP values with genetic similarity values based on microsatellite markers is low (r = 0.285, p < 0.05). The results indicate a need to develop wheat varieties with a diverse genetic background and to incorporate new variability into the existing wheat gene pool.     

Genetic Stability of Progeny from an Artificial Allotetraploid Carp Using Sperm from Five Fish Species

Microsatellite markers and D-loop sequences of mtDNA from a female allotetraploid parent carp and her progenies of generations 1 and 2 induced by sperm of five distant fish species were analyzed. Eleven microsatellite markers were used to identify 48 alleles from the allotetraploid female. The same number of alleles (48) appeared in the first and second generations of the gynogenetic offspring, regardless of the source of the sperm used as an activator. The mtDNA D-loop analysis was performed on the female tetraploid parent, 25 gynogenetic offspring, and 5 sperm-donor species. Fourteen variable sites from the 1,018 bp sequences were observed in the offspring as compared to the female tetraploid parent. Results from D-loop sequence and microsatellite marker analysis showed exclusive maternal transmission, and no genetic information was derived from the father. Our study suggests that progenies of artificial tetraploid carp are genetically stable, which is important for genetic breeding of this tetraploid fish.     

雄性普通黄颡鱼与所保护受精卵间的亲缘关系分析

父母通过保护自己的子代来确保其繁殖的成功率.在普通黄颡鱼(Pelteobagrus fulvidraco)中,雄性普通黄颡鱼具有筑巢产卵保护后代的习性,其所保护的子代与其是否有亲缘关系是有待探讨的问题.本文利用10对微卫星分子标记鉴定12窝普通黄颡鱼受精卵与护卵鱼之间的亲缘关系,并对子代的遗传多样性进行分析.在单亲鉴定中,累积非父排除概率为0.9986,平均父权相对机会(RCP)在99.989%-99.999%之间,每个子代在10个微卫星位点上的累积PI值在2006.73-604464.07之间.同时在亲权鉴定分析中,发现3窝卵子的等位基因来自2个母亲,说明雄性黄颡鱼可以和2条雌性黄颡鱼发生交配;在遗传多样性分析中,黄颡鱼子代的平均等位基因数为11.7, 无偏观测杂合度值(Ho)在0.2473-0.9866之间,多态信息含量(PIC)值0.7096-0.8993之间.通过亲权鉴定分析,可以确认看护受精卵的雄性普通黄颡鱼与受精卵间的亲子关系.     

Application of bovine microsatellite markers for genetic diversity analysis of Swiss yak (Poephagus grunniens)

In order to assess the applicability of bovine microsatellite markers for population genetic studies in Swiss yak, 131 bovine microsatellite markers were tested on a panel of 10 animals. Efficient amplification was observed for 124 markers (94.6%) with a total of 476 alleles, of which 117 markers (94.3%) were polymorphic. The number of alleles per locus among the polymorphic markers ranged from two to nine. Seven loci (ILSTS005, BMS424B, BMS1825, BMS672, BM1314, ETH123 and BM6017) failed to amplify yak genomic DNA. Two cattle Y-chromosome specific microsatellite markers (INRA126 and BM861) amplified genomic DNA from both male and female yaks. However, two additional markers on cattle Y-chromosome (INRA124 and INRA189) amplified DNA from only males. Of the polymorphic markers, 24 microsatellites proposed by CaDBase for within- and cross-species comparisons and two additional highly polymorphic markers (MHCII and TGLA73) were used to investigate the genetic variability and the population structure of a Swiss yak herd that included 51 additional animals. The polymorphic information content ranged from 0.355 to 0.752, while observed heterozygosity (HO) ranged from 0.348 to 0.823. Furthermore, a set of 13 markers, organized into three multiplex polymerase chain reactions, was evaluated for routine parentage testing. This set provided an exclusion probability in a family of four yaks (both parents and two offspring) of 0.995. These microsatellites serve as useful tools for genetic characterization of the yak, which continues to be an important domestic livestock species.     

Use of Three Different Marker Systems to Estimate Genetic Diversity of Indian Elite Rice Varieties

Genetic diversity among 42 Indian elite rice varieties, which is important for selection of parents for conventional breeding and hybrid program, was evaluated using three different types of DNA markers and parentage analysis. Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and sequence tagged microsatellite site (STMS) markers resulted in mean heterozygosity values of 0.429, 0.675 and 0.882 over all loci, respectively, and marker index values of 2.21, 4.05 and 5.49, respectively. The three molecular marker systems together provide wider genome coverage and, therefore, would be a better indicator of the genetic relationships among the 42 elite rice cultivars than those revealed using individual molecular markers. A total of 153 bands (91%) were polymorphic out of 168 bands amplified, considering all the markers together. The average genetic similarity coefficient across all the 861 cultivar pairs was 0.70 while the average coefficient of parentage was 0.10. Cluster analysis revealed that there was a very poor correlation (correlation coefficient <0.1) between dendrograms generated using coefficients of parentage and molecular marker generated genetic similarities, which can be attributed to selection pressure, genetic drift, sampling of loci and unknown relationships among supposedly unrelated ancestors. This revised version was published online in July 2006 with corrections to the Cover Date.